Adenovirus (Ad) is a double-stranded DNA virus with a genome length of about 35-40 kb. It is known that human adenovirus is classified into 7 species (A-G) including more than 60 serotypes. Adenovirus type 3, 4, 7 and 14, belonging to species B, can cause acute respiratory diseases and even fatal pneumonia. In recent years, the emergence of type 55 adenovirus (hereinafter referred to as Ad55) which is a recombinant virus of type 11 and 14 adenoviruses, has led to a series of outbreaks of community-acquired pneumonia. Death cases were sporadically reported in these epidemics. Clinically, supportive treatment is the only strategy for Ad55 infection. There are still no anti-viral drugs nor prophylactic vaccines. Therefore, development of anti-Ad55 vaccines and drugs is essential for the control of Ad55 infection in general populations, especially among military recruits, young students and the like.
At present, the only adenovirus vaccine is used in US troops. This vaccine is enteric-coated capsules comprised of wild type Ad4 and Ad7 which are passaged and propagated on diploid human fetal kidney fibroblasts, and then frozen, dehydrated, and coated with cellulose lactose. The utilization of this vaccine effectively decreased the outbreaks of adenovirus epidemic in US troops. However, the used adenovirus vaccine is still of significant drawbacks. This vaccine is mainly used to prevent Ad4 and Ad7 infection, and has no exact preventive effects on Ad14 and Ad55 which are highly pathogenic. On the other hand, this vaccine is comprised of wild-type adenovirus, which may bring safety concerns because residual live viruses discharged from the intestinal tract can easily pollute the water and thus resulting in the spread of the viruses. This vaccine, therefore, cannot be applied to general population. Replication defective adenovirus vaccine with high safety and protective capacity against Ad55 is urgently needed.
Previous studies have shown that E1 gene is essential for the replication of adenovirus, whereas E3 gene antagonizes host immune responses. Adenovirus with E1 and E3 genes deleted lose the ability to replicate in immune-competent individuals and exhibit an attenuated phenotype, whereas the major surface antigens such as Hexon and Fibre are not affected. Therefore, the use of replication defective adenovirus as a vaccine can effectively enhance its safety and expand its applicable range. Replication defective adenovirus can be propagated in complementary cell lines, such as 293 and PerC6 cells, which steadily express the E1 gene of Ad5. However, for many adenoviruses, especially those from species B, deletion of E1 and E3 genes renders them difficult to propagate in these cell lines, mainly because Ad5 E1B 55K cannot interact with E4 Orf6 from species B adenovirus, and thereby cannot effectively inhibit host mRNA transportation out of the nucleus and enhance the expression of viral late proteins.
Replication defective Ad55 can also be widely used as gene vector in gene therapy, as well as vaccines and other fields. Adenovirus vector has a number of advantages such as good safety, effectiveness of gene transduction and convenience of large-scale production. Due to these advantages, adenovirus vector has been used in hundreds of clinical trials around the world, ranking among the most widely used vectors (24.8%). Most studies use Ad5 or Ad2 as vector. However, the pre-existing anti-Ad immune response elicited by previous adenovirus infection limits the use of traditional adenovirus vector. Studies have shown that pre-existing Ad2 and Ad5 neutralizing antibodies are of high seropositive rate in developing countries and regions such as Africa, South America and China. In some populations the seropositive rate is even more than 90%. These neutralizing antibodies inhibit the entry of adenovirus vectors into body cells, making it difficult to perform immunological or therapeutic functions. To overcome the pre-existing anti-Ad immune response, researchers have developed a series of techniques, including: 1) using immunosuppressive agents to inhibit the anti-Ad immune response such as cyclosporine, cyclophosphamide, FK506, etc.; 2) modifying or reconstructing surface proteins of adenovirus vector in order to bypass pre-existing neutralizing antibodies; 3) infecting PBMC by adenovirus in vitro, then performing autologous transfusion (an AVIP techniques which we have previously developed), and so on. However, these techniques either have severe side effects (such as immunosuppressive agents) or can only be used for once because of the immune response triggered against a new vector after the application.